Anti ck 18

Protein extraction and immunoblotting were performed as previously described [47 (link)]. Primary antibodies were as follows: anti-Notch1 (Clone A6 Novus Biologicals, Cambridge, UK), anti-Thbs1(sc-73158, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-Cadherin (Clone NCH-38, Dako, Denmark) anti-mTor (2993, Cell Signaling Technology, Beverly, MA), anti-Icam5 (Abcam, Cambridge, UK), anti Pai3 (sc-99153, Santa Cruz Biotechnology), anti-Vimentin (Clone V9, Dako), anti Ck19 (Clone RCK108, Dako), anti-Ck18 (Clone DC10, Dako), anti-Mmp-9 (Clone 6-6B, Calbiochem, San Diego, USA), anti-Snail (sc-28199, Santa Cruz Biotechnology), anti-Ck8 (sc-52324, Santa Cruz Biotechnology), anti-Alpha-Sma (Clone 1A4, Sigma), anti-E-Cadherin (Clone 4A2, Cell Signaling) and anti-β-Actin monoclonal antibody (Clone AC-40). Immunoreactivities were revealed with the EnVision dextran polymer visualization system (Dako). Membranes were washed and autoradiographies were obtained using a chemiluminescence reaction (ECL reagents, Amersham). Digital images of autoradiographies were acquired with a scanner (Fluor-S MultiImager, Bio-Rad) and signals were acquired in the linear range of the scanner and quantified using specific densitometric software (QUANTITY-ONE, Bio-Rad) in absorbance units.

Immunocytochemical staining was done using the standard protocols. Cultured cells were fixed in 4% paraformaldehyde (Dingguo Biotech, Beijing, China) for 20 min and rinsed with PBS for three times. They were permeabilized with 0.5% Triton X-100 (Dingguo Biotech) for 10 min at room temperature, and then incubated in 3% hydrogen peroxide (Dingguo Biotech) for 30 min to quench the endogenous peroxidase activities. The cells were incubated with mouse monoclonal anti-albumin (1:100, Dako, Glostrup, Denmark), anti-AFP (1:100, Dako), anti-CK-18 (1:200, Dako), anti-HBsAg (1:100, Biolegend), anti-HBcAg (1:100, Biolegend) for 4 h at 37 °C, and then rinsed with PBS five times. They were incubated with anti-mouse IgG (1:1000, Biolegend) for 30 min. Immunoreactivity was visualized utilizing 3,3-diaminobenzidine tetrahydrochloride (DAB, Zhongshanjinjiao Biotech, Beijing, China) and counterstained with hematoxylin (HE, Baso) for 5 min, and then observed under a light microscope (Olympus).

Western blotting method was in accordance with the standard protocols. We used mouse monoclonal anti-albumin (1:100), anti-AFP (1:200), anti-CK-18 (1:100), anti-HBsAg (1:100) anti-HBcAg (1:100) and anti-β-actin (1:500, Dako). We used goat antibodies to mouse IgG (1:500) and developed the blots by DAB.